Stem cell-mediated therapy entails nuclear reprogramming, the alteration of gene expression patterns unique to differentiated cell types in diverse tissues that enable the stem cell to rescue or replace defective tissues. The finding that hematopoietic stem cell (HSC) derivatives can incorporate into other tissues has been well documented by expression of GFP and other markers, and has forced a reassessment of the utility of these adult stem cells and their derivatives in a range of settings. The current grant proposal focuses on the capacity of adult HSCs to incorporate into skeletal muscle and activate muscle genes. This reprogramming of HSCs remains poorly understood with respect to efficiency, mechanism, and function. HSC derivatives may first fuse with pre-existing muscle fibers followed by nuclear reprogramming in response to intracellular cytoplasmic factors. Alternatively, nuclear reprogramming could occur in response to extracellular signals in the microenvironment. Experiments proposed in Specific Aim (1) will examine and quantify reprogramming that occurs in the nuclei of HSC derivatives, in vivo, via either mechanism after transplant of HSCs into mutant mice that are null for early and late muscle gene products. In Specific Aim (2) the molecular mechanisms underlying muscle gene activation and chromatin remodeling in HSCs and other cells will be examined. The recent discovery that HSC derivatives form heterokaryons naturally in vivo warrants mechanistic studies in vitro. Loss of function studies will benefit from the use of siRNAs and the extent of nuclear reprogramming will be assessed on a gene-by-gene as well as array based approaches. In addition, reprogramming of HSCs will be examined in the absence of fusion by overexpression of early myogenic regulators of the paired box and basic helix loop helix families, or exposure to microenvironmental cues. In Specific Aim (3) loss of function genome-wide siRNA library screens will be used to identify new regulators involved in the myogenic program by taking advantage of a novel technology recently developed in our laboratory, Restriction Enzyme Generated siRNAs (REGS). The ultimate utility of HSCs will depend on their ability, either innate or after manipulation, to be reprogrammed to reliably and stably express a new set of genes, the products of which perform a function or contribute to a structure that is lacking in a particular disorder. The results of the proposed experiments may suggest ways to modulate HSC reprogramming for useful therapeutic application to enhance tissue regeneration in conditions associated with disease or aging.